Repair of ruptured membrane by injection of naturally occurring protein in amniotic fluid sac

ABSTRACT

The present invention provides compositions and methods for use in treatment of premature rupture of membrane. The compositions of the invention include a therapeutic amount of an avian thick egg white composition. Preferably, the compositions include the purified thick egg white protein, ovomucin. The methods of the invention involve injecting an effective amount of the compositions into the prematurely ruptured amniotic sac of a patient in order to seal the rupture.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of obstetricsand/or perinatal medicine. More particularly the invention relates torepair of pre-parturition rupture of the amniotic fluid sac.

2. Description of Related Art

Premature rupture of membranes (PROM) during the second and early thirdtrimester of human pregnancy creates a management dilemma forobstetricians. There are currently many management protocols for PROM.Recent literature argues the risks and benefits of tocolytic agents,antibiotics, and corticosteroid injections primarily for delayingdelivery, preventing intraamniotic infection, and enhancing fetalmaturity, respectively, in the event of almost certain preterm delivery.However, to date, no true accepted treatment for PROM exists. Medlinesearches of this topic reveal only scant data in Italian studies onintracervical instillation of fibrin glue, and Japanese attempts atmechanical blockage of the cervix using double balloon-tipped catheters.

The ideal therapy for PROM in the absence of chorioamnionitis, anddeciduitis (infections implicated in premature rupture and pretermlabor) would seem to be to recreate an intact amniotic fluid sac using abenign sealant injected into the amniotic sac using needles no biggerthan those now commonly used for amniocentesis. By recreating theintegrity of the amnion, such a technique would provide a barrier toascending infection from the normal bacterial flora of the cervix andvagina commonly isolated in most all obstetric and gynecologicinfections. Such a technique would also allow reaccumulation of normalamniotic fluid volumes, thus protecting the fetus from compression ofits own umbilical cord.

Chicken egg white is a cross species analog to amniotic fluid, providingprotection, cushioning, and nutritive substances essential to thesurvival of the unborn chick. The cross linking of this colloidsubstance at the site of membrane rupture in the human gestation mayprovide an adequate sealant in the event of PROM. The inherentproperties of egg white colloid include immiscibility with water(hydrophobic), thus creating a bolus effect when encountering theleakage site after injection into a water filled cavity. The mostgelatinous portion of the thick egg white component is of substantiallylow enough viscosity to allow injection via a standard 18 to 20 gaugespinal needle commonly used for amniocentesis.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of the priorart by providing methods and compositions for treating premature ruptureof membranes (PROM) during pregnancy. The methods and compositionsprovided result in prolonged gestation, decreased risk of infection andumbilical cord compression, and an increased likelihood of a maturefetus.

In one aspect, the invention provides a method of sealing a rupturedamniotic membrane by introducing an effective amount of an avian thickegg white composition into an amniotic fluid sac of a mammal. It isenvisioned that the thick egg white from any type of avian egg will beeffective in the present invention. The preferred composition willinclude chicken thick egg white. For administration of the into theamniotic fluid sac of a mammal, it is preferred that the compositionadditionally include any known pharmaceutically acceptable carrier. Itis not believed that the pharmaceutically acceptable carrier isabsolutely necessary in the method of the invention, however.

It is believed that the component of thick egg white that is mostimportant in accomplishing the objectives of the invention is theprotein known as ovomucin. Therefore, in preferred embodiments, thecomposition for use in the method of the invention includes isolated andpurified ovomucin or an ovomucin composition. It is envisioned that theovomucin in the composition of the invention may be present inconcentrations of about 1 to 90 percent by weight. It will be understoodthat this range of concentrations includes all integers and fractionscontained between the range of 1 to 90 percent. That is, the range ismeant to include concentrations of 1, 2, 3, 4 . . . 10, 11, 12, 13, 14,. . . 20, 21, 22, 23, 24, . . . 30, 31, 32, 33, 34, . . . 50, 51, 52,53, 54, . . . 80, 81, 82, 83, 84, . . . and so on, including fractionsof a percentage, such as 1.1, 1.2, 1.3, . . . 2.1, 2.2, 2.3, . . . 5.1,5.2, 5.3, . . . 10.1, 10.2, 10.3. . . and so on up to about 90 percentby weight.

In certain preferred embodiments, the thick egg white compositionfurther comprises one or more antibiotics.

The thick egg white composition for use in the method of the inventionwill typically be introduced into the amniotic sac by an injectionprocedure, such as an amnioinfusion procedure.

Another aspect of the invention provides a method of enhancing theintegrity of a ruptured amniotic membrane by contacting the rupturedamniotic membrane with an effective amount of an avian egg whiteglycoprotein composition. Preferably, the avian egg white compositionwill be a chicken thick egg white composition. Most preferably, thethick egg white composition will include a pharmaceutically acceptablecarrier. The thick egg white composition will generally comprise anovomucin composition where the ovomucin will be isolated substantiallyaway from other thick egg white components and purified. The ovomucinmay be present in concentrations of 1 to about 90 percent by weight. Incertain preferred embodiments, the thick egg white composition willfurther include one or more antibiotics.

It is envisioned that the thick egg white composition will typically beintroduced into the amniotic sac of a patient using an injectionprocedure. Preferably, the injection procedure will be an amnioinfusionprocedure, such as amniocentesis.

Another embodiment of the present invention provides a pharmaceuticalamniotic membrane sealant composition comprising an effective amount ofa sterile avian egg white glycoprotein composition and one or morepharmaceutically acceptable preservatives. The composition of theinvention may further include a pharmaceutically acceptable carrierand/or at least one antimicrobial agent in a therapeutically effectiveamount.

The present invention further provides a kit for use in a method ofsealing a ruptured amniotic membrane including a thick egg whiteglycoprotein composition in a suitable vial or container. Preferably,the thick egg white composition will include isolated and purifiedovomucin with a purity of about 80% to about 95%. The egg whitecomposition may be present in the vial in powder form or gel form or ina solution including a pharmaceutically acceptable preservative and/or apharmaceutically acceptable carrier. In certain aspects, such as whenthe composition is in powder or gel form, a pharmaceutically acceptablecarrier may be added directly to the vial, mixed gently, drawn into anappropriate needle for injection, and injected into a patient having aruptured amniotic membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A Thick egg white injected into water filled balloon via 20Gspinal needle.

FIG. 1B Successful occlusion of 20G holes in balloon by thick egg white.

FIG. 2 Condom-balloon apparatus simulating uterine lining (condom) andamnion (balloon). Successful occlusion using thick egg white.

FIG. 3 Physiologic in vitro evaluation of thick egg white occluding six1.2 mm (18G) holes in human amnion. Saline solution was utilized torecreate a resting uterine tone of 12 cm water pressure.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Premature rupture of membranes (PROM) during pregnancy is currentlyattributed primarily to infection preceding rupture. More specifically,PROM is caused by rhexis of the amnion, especially in the area incontact with the uterine cervix. Rhexus of the amnion may be cause by(1) physical weakening of the amnion, (2) elevation of intrauterinepressure, and (3) dilation of the cervix. Infection may accompany eachof these conditions, all of which interact with each other.

It has been discovered that amniotic fluid may not be sterile, contraryto previous beliefs. For example, Miller et al. (1980) showed thatbacterial colonization of amniotic fluid might exist despite intactmembranes, suggesting that bacteria can pass through the fetal membrane.Galask et al. (1984) demonstrated in vitro that anaerobes and group Bstreptococci (GBS) could easily invade the membrane.

Subclinical chorioamnionitis is believed to be a cause of idiopathicpremature labor. Many genital bacteria are known to produce bothphospholipase A₂ and phospholipase C, which can stimulate increasedrelease of arachidonic acid and prostaglandin production within theuterus. Bejar et al. (1981) have shown that some genital bacteriarelease a phospholipase enzyme, thereby activating arachidonic acidmetabolism in fetal membranes and stimulating the biochemical componentsof labor. Huddleston (1982) argues that the release of phospholipase A₂from the fetal membrane triggers labor and leads to prostaglandinsynthesis in the placental membrane.

Although the fetal membrane is normally very strong, clearly it ispossible for it to become mechanically fragile. The only reasonableexplanation for weakening of the membrane is colonization with vaginalflora. Bacterial invasion of the fetal membrane causes localinflammation, i.e., focal chorioamnionitis, with loss of membraneintegrity leading to PROM. Bacterial enzymes and host products secretedin response to infection may add to the weakening and rupture of themembranes.

Resistance to infection depends on natural physiologic and mechanicalbarriers such as the endocervical mucus plug, intact fetal membranes andantimicrobial properties of amniotic fluid. PROM destroys thosebarriers. If PROM is left untreated, labor ensues, and the fetus isdelivered with premature function of various organs. For example, when ababy is born with premature pulmonary function, he is likely to developrespiratory distress syndrome (RDS). Severe prematurity and immatureepithelial lining of the germinal matrix in the brain or of the liningof the inter intestines can predispose the neonate to intraventricularhemorrhage (IVH) and necrotizing enterocoliter (NEC), respectively.Therefore, it is desirable to prolong the gestation of the fetus.

Typically, a patient who has suffered premature membrane rupture isimmobilized in bed and sometimes given a tocolytic agent and anantibiotic. This treatment has often not proved sufficient to preventmaternal and fetal infections or labor, thus still often resulting inpremature birth. Thus, while the infusion of antibiotics and otheragents may be helpful, it would be more effective to provide some sortof physical plug or barrier to ascending infection. Such a barrier wouldalso prevent the leakage of amniotic fluid out of the amniotic sac andaid in prolonging the gestation.

The inventor has discovered that the gelatinous nature of avian eggwhite and proteins within it are effective to create a seal of amembrane rupture when injected into the amniotic sac. While it isbelieved that the white from virtually any avian egg will be effectivein the methods of the present invention, the examples herein focusprimarily on the chicken egg white.

Chicken egg white is a cross species analog to amniotic fluid, providingprotection, cushioning, and nutritive substances essential to thesurvival of the unborn chick. The cross linking of this colloidsubstance at the site of membrane rupture in the human gestation mayprovide an adequate sealant in the event of PROM. The inherentproperties of egg white colloid include immiscibility with water(hydrophobic), thus creating a bolus effect when encountering theleakage site after injection into a water filled cavity. The mostgelatinous portion of the thick egg white component is of substantiallylow enough viscosity to allow injection via a standard 18 to 20 gaugespinal needle commonly used for amniocentesis.

Chicken egg white exists as layers of thick egg white and thin eggwhite. The total egg white is more than 50% thick egg white, which isanchored by the chalazae to the surface of the yolk membrane. Thechalazae and at least part of the thick egg white are closely relatedphysically and probably chemically. In fact, the chalazae cannot beremoved from the egg without tearing it from the egg white. The fibersof chalazae appear to become finer and finer until they admix and“disappear” into the thick egg white.

Thick and thin egg white differ both physically and chemically. Physicalseparation of the thick and thin egg white is currently only possible bycrude and approximate means. The most simple and obvious method is tocut the thin and thick white away from one another in the broken-outegg. Another commonly used method is to separate the egg whites by meansof passing the egg white through sieves with large diameter holes.Ovomucin has been purified to about 90% purity by French scientistsusing a Sepharose gel electrophoresis process.

The chemical differences between the thick and thin egg white includethe following: When egg white is diluted and acidified, approximatelyfour times the amount of precipitate is obtained from thick egg white asfrom thin egg white. This precipitate is called crude ovomucin. Ovomucinis believed to be the component responsible for the sealant propertiesof the egg white in the present invention. Additionally, thick egg whitecontains approximately four times the amount of inhibitory activityagainst the hemagglutination of erythrocytes by viruses. Thick egg whitealso contains approximately 30-40% more N-acetyl-neuraminic acid (sialicacid) than does thin egg white.

Chalazae, extracts of crude yolk membranes, and ovomucin also possesscertain similarities between them. For example, all three are relativelyhigh in sialic acid and in inhibitory activity for viralhemagglutination. These similarities support the hypothesis that thefibers in thick egg white, the chalazae, and the contiguous layer ofthick egg white which is intimately associated with the true yolkmembrane are closely related, if not identical, substances. Theseobservations are also consistent with Conrad and Phillips' hypothesisthat the chalazae is formed from the ovomucin fibers in the thick eggwhite as the egg passes down the hen's oviduct. Conrad and Phillips(1938) speculate that while the entire egg rotates slowly during itsoviductal passage, the yolk remains steady and does not rotate with therest of the egg. As a consequence, the ovomucin fibers are slowly turnedand twisted into what eventually becomes the chalazae. There are,however, no direct observations to substantiate this attractivemechanical theory for the formation of chalazae.

The egg white is known to be primarily a solution of proteins with arelatively small amount of sugar and salts. Hence, the biochemistgenerally prefers to use avian egg white when he desires to separate,fractionate, and prepare purified proteins in quantity. That the eggwhite is primarily a solution of proteins makes the job of proteinseparation and purification much easier than with many other biologicalmaterials. Indeed, several egg white proteins have become standardproteins for biochemists. For example, chicken ovalbumin and lysozymehave been two of the less expensive and highest purity proteinsavailable for many years.

The primary protein of chicken egg white is ovalbumin. The concentrationof ovalbumin in egg white is about four to five times the concentrationof the secondary constituents, ovotransferrin, and ovomucoid. Thus,ovalbumin provides the primary properties of egg white with the otherconstituents contributing mainly to the biological properties of the eggwhite. Ovumucin is an exception to this general rule. Ovomucin appearsresponsible for the high viscosity of thick egg white.

Many different procedures are available for fractionating chicken eggwhite for the preparation of the individual proteins. These proceduresare well within the skill or the ordinary artisan and include saltfractionation techniques and fractionation with cellulose ion-exchangeagents. Additionally, serial acidification, precipitation,centrifugation and alkali resuspension can be used to separate andpurify ovomucin.

PROPERTIES OF EGG-WHITE PROTEINS

Ovalbumin is the principal protein of chicken egg white and is also theonly major constituent for which no unique biochemical activity has beenfound. The main property of ovalbumin has been its sensitivity todenaturation. Ovalbumin is known to have some interesting chemicalaspects not directly related to sequence. One such interesting aspect isits heterogeneity due to the presence of either one or two phosphates.Another interesting aspect is the number and reactivity of the cysteinesulfhydryl groups. Yet another is the structures of the side-chaincarbohydrates and linkages of the carbohydrates to the peptide chain.

Avidin is another protein prominent in egg white. In 1941 Eakin, Snell,and Williams concentrated avidin using acetone and salt fractionationprocedures. They then performed a modified biotin microbial assaydesigned for avidin. A unit was defined as the amount of concentratecapable of inactivating 1 gram of biotin. Raw egg white varied from 0.8to 1.2 units/cc. Free avidin appears to be an albumin-like protein,although it probably occurred in egg white to varying extents as theabove mentioned complexes.

Chicken egg-white flavoprotein (or ovoflavoprotein) is one of twoegg-white proteins which contains, or can bind, a B-vitamin. The otheris avidin. Rhodes, Bennett, and Feeney (1959) found ovoflavoprotein toexist in two forms; one form, flavoprotein, contains riboflavin and theother, apoprotein, does not. The total concentration of the two forms isapproximately 0.8% of the egg white.

Ovomacroglobulin is the only detectable component of avian egg whites tohave a wide spectrum of immunological cross-reactivity. It also appearsto be strongly immunogenic. Deutsch (1953) reported that a large part ofthe capacity of egg white to produce antibodies was probably a minorunrecognized fraction of the egg white. The ovomacroglobulin might bethis fraction.

Ovomacroglobulin is a very large protein with an estimated molecularweight of approximately 800,000 g/mole as determined bysedimentation-velocity, diffusion, and light scattering measurements.This makes ovomacroglobulin the largest recognized egg-white protein,other than ovomucin. There is, however, evidence that it dissociatesinto smaller units in acid solution.

In addition to the major proteins already described, there are probablyone or two dozen minor proteins in chicken egg white. Several of thesehave been seen in different types of electrophoretic patterns of the eggwhite itself and probably many more have been seen as minorcontaminating substances on purification of the recognized constituents.Lineweaver and co-workers (1948) found chicken egg white to beessentially devoid of enzymatic activities with the exception ofcatalase activity.

Some of the egg white proteins are known to have antimicrobial activity.It is believed that those proteins that do show antimicrobial orantienzyme properties could potentially be used as microbialantagonists. These include lysozyme, ovoflavoprotein, avidin,ovotransferrin, and the inhibitors of proteolytic enzymes, ovomucoid,and ovoinhibitor. For example, lysozyme has been shown to bebactericidal to a limited number of species. Ovotransferrin has alsoproven to be an important antimicrobial substance. Egg white typicallyhas a high pH during the first few days of the incubation of the egg.This high pH egg white also has antibacterial activity, a factor that isusually overlooked.

The pH of the egg white in a freshly laid chicken egg is slightly below7.6 and the pH of the yolk is approximately 6.0. The initial pH of 7.6obviously closely approximates the pH of blood serum. Since the eggshell contains approximately ten thousand pores, carbon dioxide escapesthrough the shell and the pH of the egg white begins increasingimmediately after the egg is laid. The rate of the pH increase dependson the temperature and the carbon dioxide tension. The pH will rise toabove 8.0 within a matter of three or four hours at room temperatures.It appears that the egg white has such a high carbon dioxide contentbecause a high carbon dioxide tension exists in the oviduct.

Physical changes in the egg contents occur after a few days at roomtemperature or a few weeks at refrigerated temperature. These changesinclude a weakening and dissolution of the gel of the thick egg whiteand a weakening and eventual rupturing of the yolk membrane. The rate atwhich these changes occur depends on the pH of the egg white and thetemperature of incubation. The physical changes themselves are wellknown to anyone who has broken out a partially deteriorated egg andobserved the watery white and perhaps rupturing of the yolk membrane.For this reason, it is believed that the egg white should be used withinabout 2 days if maintained at room temperature, preferably within about24 hours. The composition could typically be usable for up to 2 weeksafter purification and resuspension in a pharmaceutically acceptablecarrier. Most preferably, the composition will be maintained at bodytemperature, that is, at about 37° C. and will be used within about 24hours.

Most investigators agree that the deteriorative process in the thick eggwhite involves changes of the ovomucin. It has been shown that egg whitecan be thinned and the yolk membrane weakened by adding small amounts ofreducing agents such as mercaptans or sulfite. (Hoover 1940; MacDonnellet al. 1955). Several treatments that denature or inactivate proteinprevented the changes. It was further shown that other deteriorativechanges in egg white were most evident by changes in theovotransferrins. These changes were apparently due to interactions ofthe proteins with the glucose naturally present in the egg white. Thisglucose-protein reaction (Millard) seems to be accelerated by alkalineconditions and egg white is known to be relatively alkaline. Such aglucose-protein reaction should not occur at room temperature (or 37°C.) and in relatively dilute solution.

Several theories have been proposed for the deteriorative mechanisms inegg white. The first theory is that ovomucin is reductively cleaved byreducing agents generated during the incubation of the white. It isknown that ovalbumin, which contains sulfhydryl groups, can be titratedwith p-chloromercuricbenzoate. (MacDonnell et al. 1955). A“denaturation” of less than 1% of the ovalbumin is likely enough tosupply the potential amount of reducing agent required to thin the eggwhite. Changes have been reported in the chromatographiccharacteristics, in the solubility, and in resistance to denaturation ofovalbumin.

The second theory is that lysozyme and ovomucin exist in a complex thatboth forms and dissociates during incubation. This changes the gelcharacteristics and causes thinning. The formation of a complex issupported by observations that the enzymatic activity of lysozyme in eggwhite decreases during incubation. In fact, the assayable activities oflysozyme in egg white were found to drop extensively (up to 40%) duringthe thinning process. The observed activity was dependent on the ionicstrength during dilution of the egg white. These observations indicatean ionic type of complex involving lysozyme.

Another theory is that the solubility of ovalbumin changes duringincubation. It has also been postulated that the interaction of glucosewith protein directly or indirectly causes thinning. As discussed above,glucose interaction could indirectly cause thinning by generatingreducing compounds.

Unfortunately, none of these theories fits all of the available data.Chicken, turkey (Meleagris gallopavo), and probably penguin (Pygoscelisadeliae) eggs deteriorate rapidly, while duck and goose eggs showessentially no deterioration. Duck eggs are relatively low in lysozymeand very low in sialic acid, which are both considered to be related todeterioration. The ovomucin fractions are known to contain sialic acid.Since the penguin egg contains essentially no lysozyme and the highestamount of sialic acid observed in any bird, these latter mechanisms seemimprobable. Thus, a reduction mechanism or a combination of severalmechanisms may be the cause of the changes finally expressed as a changein the colloidal distribution of the high molecular weight aggregates ofovomucin.

The inventor has found that a composition comprising avian thick eggwhite is effective to seal a membrane rupture. Since egg white is abenign composition, it is believed that injection of the purified thickegg white alone will be effective in the method of the invention.Alternatively, a composition containing ovomucin, the protein thatcauses the gelatinous nature of the egg white, may be used in theconjunction with the method of the invention.

In a preferred embodiment, the method of the invention includesadministering a composition comprising substantially purified ovomucinin a pharmaceutically acceptable carrier in a therapeutically effectiveamount to a patient who has suffered a premature rupture of membrane.The term “substantially purified” refers to a ovomucin that has beenisolated away from the other proteins present in thick egg white andpurified to at least about 80% purity. It may also include syntheticovomucin prepared to at least about 90% purity. Preferably, the isolateand purified ovomucin, either synthetic or natural, will be prepared toabout 98% purity. It is also envisioned that active fractions orepitopes of the ovomucin protein may be effective in the presentinvention. Protein analysis to locate active epitopes and activeportions of the protein chain, and preparation of peptide fractions, ortruncated proteins, are well known and routine to those skilled in theart.

In preferred embodiments the composition may also contain one or moreantibiotic or antimicrobial agents. For example, antimicrobials whichwill be effective in the present invention include injectablepenicillins or their derivatives, injectable cephalosporins or theirderivatives, injectable microlide antibiotics or their derivatives,injectable aminoglycoside antibiotics or their derivatives or othernaturally occurring bacteriocidal and/or bacteriastatic substances foundin egg white matrix. The antimicrobials will be present in thecomposition in a therapeutically effective amount, that is, in an amounteffective to eliminate or inhibit bacteria present in the amniotic sacdue to ascending infection or otherwise. The antimicrobials forinclusion in the compositions of the invention will generally be presentin concentrations that provide bacteriocidal activity based on a minimuminhibitory concentration in the presence of a maximum dilution of about3,000 mL of amniotic fluid.

The compositions of the invention will be injected directly into theruptured amniotic sac of a patient who has suffered from prematurerupture of membranes (PROM). Preferably, the compositions of theinvention will be injected using an 18 gauge or a 20 gauge spinalneedle, although it is envisioned that other sized needles, such as 18to 25 gauge needles of variable length may be used in conjunction withthe method of the invention. Preferably, about 5 to about 20 ccs will beinjected into the patient's amniotic sac. Most preferably, about 10 ccswill be injected into the patient who has suffered from prematurerupture of membrane.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all such cases, the form must be sterile and must befluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thiomerisal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecomposition of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously-sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

EXAMPLE 1

Nine inch diameter helium grade water filled balloons were initiallyused as an in vitro model of the amniotic membrane. The internalpressure as measured by manometry was between 25-35 cm of water.Hypodermic needles of standard medical grade were used to createpuncture holes in the water filled balloons. Initially, single 20 gauge(1.0 mm) holes were created through which 5 mL of thick egg white wereinjected into the water filled balloons (See FIG. 1A). The hydrophobicegg white remained immiscible with water. When the egg white boluscontacted the inner surface at the leak site, the hole was successfullyplugged (FIG. 1B). These seals were maintained for three weeks, at whichtime, the integrity of the rubber of the balloon itself began todegrade. Of note, the seal appeared more readily when the water insidethe balloon was approximately equal to body temperature (37° C.) asregulated by a digital thermometer.

EXAMPLE 2

Repeated acetic acid elutions were performed on thick egg white.Repeated elutions with 5% acetic acid followed by washing with water ledto thickened and precipitated membrane in the thick egg white. The thinegg white was easily separated from the thick egg white in the solubleform. Resuspension occurred when the pH was made basic using ammonia.Precipitation again occurred when acidic conditions were reinstated.Thus, it appears that the components of thick egg white are stable tochanges in pH of the suspending solution, but the viscosity is markedlyincreased in acidic environments.

EXAMPLE 3

A dilute (5%) acetic acid was used to thicken and precipitate the thickegg white. The more viscous component was separated from the remainingegg white components. A nine-inch helium-grade balloon was then filledwith water and 4 mL of acid eluted thick egg white. The balloon (innermembrane) was then placed inside a slightly water filled latex condom.This apparatus simulates the inner membrane (amnion) and outercompartment (uterus). A single 18 gauge (1.2 mm) hole was created bypassing a hypodermic needle through the balloon. The thick egg whitewithin the balloon migrated to the leak site and readily sealed the leakat the balloon/condom interface (FIG. 2). The seal was readilyvisualized through the transparent condom.

EXAMPLE 4

Amniotic membranes from human placentas were utilized to more accuratelyassess in vivo activity of the thick egg white physical characteristics.Membranes from normal term placentas were attached to the distal end ofa 12-inch long 1 ½ inch diameter common polyvinyl chloride (PVC) pipe. Asingle rubber band was used to secure a 12 cm×12 cm square of amnion tothe PVC pipe. Normal saline solution (0.9%) was used to fill theproximal end of the pipe to the gravitational equivalent pressure to theresting tone of a pregnant uterus. Approximately 8 mL of thick egg whitewas introduced into the proximal end of the pipe. Six 18-gauge (1.2 mm)holes were punctured through the amniotic membrane (FIG. 3). All holeswere readily sealed using the above apparatus.

The same apparatus was utilized while creating a single 1 cm tear in theamnion. The saline and egg white solution failed to seal this size tear.It is believed that the leakage through such a tear in an actual humanamniotic membrane would be lessened in vivo in a system with endometriumand outer compartment (uterus) containing the inner membrane due toincreased surface area and surface tension. The flow across the in vivosystem would be markedly less than the full gravitational forces presentin this example.

The above procedure was repeated using holes of sizes 25 gauge, 22gauge, 20 gauge and 18 gauge and a range of pressures of 10 cm, 20 cmand 30 cm of water. Some suggestion of slow leak was observed with 30 cmof water only with the 18 gauge hole. Identical results were observedusing both thick meconium and normal amniotic membrane specimenssuspended on PVC pipe.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Awade, A. C. et al., Two-step chromatographic procedure for thepurification of hen egg white ovomucin, lysozyme, ovotransferrin andovalbumin and characterization of purified proteins, J. CHROMAT. A, 677:279-288 (1994).

Awade, A. C. and Efstathiou T., Comparison of three liquidchromatographic methods for egg-white protein analysis, J. CHROMAT. B,723: 69-74 (1999).

Bejar, R. et al., Premature labor. II. Bacterial sources ofphospholipase, OBSTET. GYNECOL. 57: 479 (1981).

Conrad, R. M. and Phillips, R. E., The formation of the chalazae andinner thin white in the hen's egg, POULTRY SCI.17: 143 (1938).

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What is claimed is:
 1. A method of sealing a ruptured amniotic membranecomprising introduction of an effective amount of an avian thick eggwhite composition into an amniotic fluid sac of a mammal.
 2. The methodof claim 1, wherein said avian egg white composition is a chicken thickegg white composition.
 3. The method of claim 1, wherein said thick eggwhite composition further comprises a pharmaceutically acceptablecarrier.
 4. The method of claim 1, wherein said thick egg whitecomposition comprises an ovomucin composition.
 5. The method of claim 4,wherein said ovomucin composition comprises ovomucin in the range of 1to 90 percent by weight.
 6. The method of claim 1, wherein said thickegg white composition is introduced into said amniotic sac by aninjection procedure.
 7. The method of claim 6, wherein said injectionprocedure is an amnioinfusion procedure.
 8. The method of claim 1,wherein said thick egg white composition further comprises one or moreantibiotics, or bacterial inhibiting substances.
 9. A method ofenhancing the integrity of a ruptured amniotic membrane comprisingcontacting said ruptured amniotic membrane with an effective amount ofan avian egg white glycoprotein composition.
 10. The method of claim 1,wherein said avian egg white composition is a chicken thick egg whitecomposition.
 11. The method of claim 1, wherein said thick egg whitecomposition further comprises a pharmaceutically acceptable carrier. 12.The method of claim 1, wherein said thick egg white compositioncomprises an ovomucin composition.
 13. The method of claim 12, whereinsaid ovomucin composition comprises ovomucin in the range of 1 to 90percent by weight.
 14. The method of claim 1, wherein said thick eggwhite composition is introduced into said amniotic sac by an injectionprocedure.
 15. The method of claim 14, wherein said injection procedureis an amnioinfusion procedure.
 16. The method of claim 1, wherein saidthick egg white composition further comprises one or more antibiotics,or bacterial inhibiting substances.
 17. A pharmaceutical amnioticmembrane sealant composition comprising an effective amount of a sterileavian egg white glycoprotein composition and one or morepharmaceutically acceptable preservatives.
 18. The composition of claim17, further comprising a pharmaceutically acceptable carrier.
 19. Thecomposition of claim 18, further comprising an antimicrobial agent in atherapeutically effective amount.
 20. A kit for use in a method ofsealing a ruptured amniotic membrane, said kit comprising a thick eggwhite glycoprotein composition in a suitable vial or container.
 21. Thekit of claim 20, wherein the thick egg white composition comprisesisolated and purified ovomucin with a purity of about 80% to about 95%.22. The kit of claim 21, wherein the egg white composition may be inpowder form or gel form or in a solution comprising a pharmaceuticallyacceptable preservative with or without a pharmaceutically acceptablecarrier.